Efficient production of salicylic acid through CmeR-PcmeO biosensor-assisted multiplexing pathway optimization in Escherichia coli.

To address the challenge of microbial tolerance in industrial biomanufacturing, we developed an adaptive evolution strategy for Escherichia coli W3110 to enhance its salicylic acid (SA) tolerance. Utilizing a CmeR-PcmeO biosensor-enabled high-throughput screening system, we isolated an SA-tolerant variant (W3110K-4) that exhibited a 2.3-fold increase in tolerance (from 0.9 to 2.1 g/L) and a 2.1-fold improvement in SA production (from 283 to 588.1 mg/L). Subsequently, the designed sensors were combined with multi-pathway sgRNA arrays to dynamically modulate the other three branched-chain acid derivatives, achieving a balance between biomass growth and rapid SA production in the adaptively evolved strain, resulting in a maximum SA yield of 1477.8 mg/L, which represents a 30% improvement over the non-evolved control strain W3110K-W2 (1138.2 mg/L) using the same metabolic strategy. Whole-genome sequencing revealed that adaptive mutations in genes such as ducA* and anti-drug resistance C2 mutation genes (ymdA*, ymdB*, clsC*, csgB*, csgA*, and csgC*) play a key role in enhancing SA tolerance and productivity. Notably, the evolved strain W3110K-4 exhibits significant resistance to bacteriophages, making it a promising candidate for large-scale SA fermentation. This work develops and expands the CmeR-PcmeO system, proposes new insights into improved strains through biosensor screening, guided multi-pathway metabolism, and adaptive evolution, and provides a paradigm for engineers to obtain engineered strains.